The long-term goal of this project is to understand the molecular reactions that appear to be responsible for neuronal exocytosis, and more generally for membrane trafficking throughout eukaryotic cells. A number of proteins that participate in exocytosis have been identified, including the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP-25. These three proteins are referred to collectively as SNAREs (soluble NSF-attachment protein receptors), and are representative of families of related proteins that function at different membrane trafficking steps throughout the cell. The three SNAREs bind tightly to each other to form a SNARE complex that is thought to interconnect membranes destined to fuse with each other. SNARE complexes are stable unless dissociated by the ATPase NSF (N-ethylmaleimide sensitive fusion protein), and cycle assembly and disassembly of SNARE complexes is correlated with membrane fusion in vivo. However, it remains unclear exactly how these reactions are linked to membrane fusion. In the proposed experiments, we will determine how the SNARE complex participates in membrane fusion by generating soluble SNARE complexes, delineating their structure, and characterizing their interactions with other critical complexes in solution will lead up to predictions about how such complexes affect membranes in vivo. We ill test these predications using 'model neuronal membranes' produced by heterologously expressing the synaptic SNAREs on secretory vesicles in the yeast Saccharomyces cerevisiae, isolating these vesicles, and studying their interactions using biochemical and ultrastructural approaches. With this information we will be better able to understand how intracellular membrane fusion, and in particular neurosecretion, can be modulated or abolished and how such alterations contribute to human disease and could be exploited to therapeutic advantage.